This proposal describes plans to accelerate the development of strategies key to addressing the grand challenge of understanding structure and function within whole cell nuclei down to the nanometer scale. Project aims will target technologies for high-resolution electron dense labeling (molecular painting) of multiple specific gene loci using light-driven photooxidation of intranuclear structures and chromatin domains. This project will also improve technologies for multicolor EM we have developed to more efficiently discriminate between two electron dense regions marked with different lanthanides. We will combine these labeling methods with advantages that arise from use of direct electron detection (DD) cameras to improve energy-loss imaging by increasing our ability to use the low-loss region of the energy spectrum. Activities are organized into three aims: 1) to establish methods for introducing fluorescent labels to follow activation or inactivation of specific functional domains in the cell nucleus and use these fluorescent probes to drive the labeling of specific regions within the cell nucleus and associated chromatin; 2) to integrate the application of NCMIR developed Direct Electron Detection Cameras (DD's) with energy loss spectroscopy to increase the sensitivity of multicolor EM detection; and 3) to extend, refine and apply existing modules in NCMIR's imaging, reconstruction and analysis workflow to enhance resolution in tomographic reconstructions, develop scripts to facilitate long-exposure drift-corrected image acquisition, and add new tools for processing of images of lanthanide stained multicolor specimens. This project will be propelled by a strong Driving Biological Project comprised of synergistic activities of the laboratories of Drs. David Spector and Clodagh O'Shea, two recognized leaders in nuclear structure. Both are focused on the functional organization of the cell nucleus and aim to determine how chromatin structure is altered when specific regions are activated or inactivated by processes related to oncogenesis.